The objective of this research is to characterize the mechanisms by which the facultative intracellular pathogen, Legionella pneumophila initiates disease. In the infectious process, attachment of this organism to host cells occurs as an essential step prior to the endocytotic process and subsequent intracellular replication. The specific aim of this proposal is to probe the nature of the early steps in bacterial attachment to eukarytoic cells (macrophages) using virulent and non-virulent strains. Competitive blocking experiments as well as biochemical treatment of organisms and host cells, coupled with appropriate controls, will be used to identify, purify and partially characterize those bacterial receptors or "adhesins" involved. Such isolated microbial "adhesins" will be investigated per se for their ability to block or prevent infection of cells in vitro. Adherence or inhibition of attachment will be assayed by organism recovery studies (viable colony counts), immunofluorescence, radiolabeling and ELISA techniques. Furthermore, the mechanisms involved in bacterial adherence as determined by biochemical means will be monitored by transmission and scanning electron microscopy. Antimicrobial chemotherapeutic agents will be used as a tool to evaluate the role of bacterial protein synthesis in the expression of such bacterial virulence factors as "adhesins". From this, their influence on the specific binding steps and thereby the initial stages of the disease process will be examined. The overall goal of this work is to probe the earliest stages of the infectious process at the cellular and molecular levels and thereby obtain fundamental data on the intracellular strategy of this human pathogen. Detailed investigations on the nature of bacterial adherence, which forms a necessary prelude to bacterial endocytosis, intracellular replication and eventually the disease state, will provide potential and novel methods for control measures against this organism and the prevention of clinical legionellosis.